Class of phosphocholine derivatives having antifungal activity

ABSTRACT

Certain phosphocholine derivatives having substantial antifungal therapeutic activity are disclosed. The phosphocholine derivatives may be chemically synthesized, enzymatically prepared or extracted from the plant Irlbachia alata. The phosphocholine derivatives are useful in treating fungal infections including those which are dermatophytic, systemic, ophthalmic and vaginal.

This is a continuation-in-part application of U.S. patent applicationSer. No. 07/958,416, filed Oct. 8, 1992, now abandoned, the entiredisclosure of which is incorporated by reference.

1. FIELD OF THE INVENTION

This invention relates to new classes of phosphocholine derivatives aswell as to various methods for preparing these compounds--includingsynthetic, enzymatic and extractive using certain plants. Thephosphocholine derivatives of the invention are non-toxic and exhibitsubstantial antifungal activity in slowing fungal growth and in killingfungi.

2. BACKGROUND OF THE INVENTION

The plant species Irlbachia alata has been used as an anti-infectiveagent in the Peruvian Amazon region. The leaves are squeezed and theliquid is applied to infected skin sores. The same liquid from theleaves is applied to skin problems and skin fungal infections. It isutilized to treat vaginal yeast infections.

Irlbachia alata is one species of 10-12 species of the plant familyGentianaceae. These species occur in tropical South America especiallyin the Amazon and Negro River basins. The plants in the genus Irlbachiaare generally low herbs characteristically with 3-5 plinerved leaves.The most consistent diagnostic feature for the genus is the pollenmorphology.

A reference to Irlbachia alata and related species was made in 1775 bythe French scientist Fusee Aublet (Aublet, F. 1775, Histoire des Plantesde la Guiane Francoise, Didot, Paris). The ethnobotanical notes fromthis reference were subsequently compiled and republished in English.Aublet noted the following about two species in the genus Irlbachia:

Irlbachia Alata The entire plant is bitter. It is used to clearobstructions; I (Aublet) have used it with good results. The species iscalled "Bois creux" (Hollow wood) by the Creoles.

Irlbachia Pururascens All parts of this plant are bitter. It is used asan apertif and to reduce fever.

3. SUMMARY OF THE INVENTION

We have discovered a class of phosphocholine derivatives (Class I)having extraordinary antifungal activity.

Structurally, these compounds are phosphocholine derivatives (1 or2-deacyl-phosphatidyl cholines) in which the 1 or 2-OH-group of theglycerol moiety has been glycosylated with glucose, galactose,arabinose, mannose, rhamnose or another sugar. The basic chemicalstructure may be drawn as follows: ##STR1## wherein one of R or R' is asugar moiety and the other is an acyl or sugar moiety.

The molecular backbone common to all members of this class of compoundsis drawn above. The acyl-group can be any long-chain fatty acid, whilethe sugar unit can be any of the sugars commonly found in plants,including but not limited to glucose, galactose, arabinose, mannose,rhamnose, or another naturally occurring sugar.

We have additionally found a structurally related class ofphosphocholine derivatives of similar or greater antifungal activitythan the above-discussed class of phosphocholine derivatives (i.e.,Class I).

One novel class of phosphocholine derivatives (Class II) havingantifungal activity has the basic structure shown below: ##STR2## whereQ is C2 to C30 alkyl, alkenyl, alkynyl, branched alkyl, branchedalkenyl, or branched alkynyl;

Z is oxygen or sulfur; X and Y are independent oxygen, sulfur, CH₂, CF₂,or N-R₁ ;

A, B, and T are independently alkyl, alkenyl, alkynyl, branched alkyl,branched alkenyl, or branched alkynyl radicals of C1 to C20 chainlengths; are independently or together cycloalkyl or bridged cycloalkylradicals of ring size C3 to C20, or cylcoalkenyl, bridged cycloalkenylor cyclo(polyene)radicals of ring size C4 to C20, cycloalkynyl, bridgedcycloalkeynl or cyclo(polyalkynyl)radicals of ring size C8 to C20;

D is oxygen, sulfur, CH₂, CF₂, or N-R₂ ;

F is alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branchedalkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl or cycloalkynylradicals containing C1 to C20 carbon atoms;

R₁ and R₂ are independently hydrogen, alkyl, alkenyl, alkynyl, branchedalkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridgedcycloalkyl, cycloalkenyl, bridged cycloalkenyl or cycloalkynyl radicalscontaining C1 to C20 carbon atoms, or any protecting group described inthe book "Protecting Groups in Organic Synthesis" by Theodora Greene andPeter G. M. Wuts.

Another class of phosphocholine derivatives (Class III) havingantifungal activity has the following structures: ##STR3## where AA, BB,DD are independent of each other, equal to each other, or interchangedas shown above, the central carbon atom can be either the R and Soptical stereoisomer or a mixture of R and S stereoisomers, and whereAA, BB, and CC DD defined as follows:

where AA, is A-J with A being attached to the carbon atom of the threecarbon central unit and J is defined below;

BB is B-Y, with B being attached to the carbon atom of the three carboncentral unit and Y is defined below:

DD is ##STR4## where W, E, G and Q are defined below; A is oxygen,sulfur, CH₂, CF₂ or N-R₁ ;

B is oxygen, sulfur, CH₂, CF₂ or N-R₂ ;

D is oxygen, sulfur, CH₂, CF₂ or N-R₃ ;

Y is alkyl, alkenyl, alkynyl, poly(alkenyl), poly(alkynyl), orpoly(alkenoalkynyl) radicals comprised of C1 to C20 carbon atoms chainlengths, or alkanoyl, alkenoyl, alkynoyl, poly(alken)oyl, poly(alkyn)oylor poly(alkenoalkyn)oyl radicals comprised of C2 to C20 chain lengths oralkyloxy, alkenyloxy, alkynyloxy, poly(alkenyl)oxy, poly(alkynyl)oxy,poly(alkenoalkynyl)oxy radicals comprised of C1 to C20 carbon atoms;

J is a furanose or pyranose radical of the type: ##STR5## where X isoxygen, sulfur, CH₂, CF₂ or N-R₄ ; F, K, L and M are independentlyhydrogen, hydroxyl, protected hydroxyl (as described in the book"Protecting Groups in Organic Synthesis" by Theodora Greene and Peter G.M. Wuts), alkyloxy, thiol, alkylthio, arylthio, alkylsulfonyl,arylsulfonyl, amino, ammonium, alkylamino, alkylammonium, dialkylamino,dialkylammonium, trialkylamino, trialkylammonium where the alkyl chainon nitrogen is comprised of C1 to C20 carbon atoms; or alkyl, alkenyl,or alkynyl radicals comprised of C1 to C20 carbon atoms.

Z is oxygen or sulfur

E is oxygen, sulfur, CH₂ CF₂ or N-R₅ ;

G is alkyl, branched alkyl, cycloalkyl or bridged cycloalkyl radicals ofC1 to C20 chain lengths;

Q is halogen, hydroxyl, protected hydroxyl utilizing any protectinggroups described in the book "Protecting Groups in Organic Synthesis" byTheodora Greene and Peter G. M. Wuts, O-arylsulfonyl-, O-alkylsulfonyl-or O-(perfluoroalkyl)sulfonyloxy, amino, ammonium, alkylamino,alkylammonium, dialkylamino, dialkylammonium, trialkylamino,trialkylammonium where the alkyl chains on nitrogen are C1 to C20, orQ=NR₁ R₂ R₃, where R₁, R₂, or R₃ can independently or together be amixture of alkyl groups of C1 to C20 in chain length and a protectinggroup described in the book "Protecting Groups in Organic Synthesis" byTheodora Green and Peter G. M. Wuts, and R₁ can equal R₂, R₂ can equalR₃, or R₁ can equal R₃ which can equal R₃ ;

R₁, R₂, R₃, R₄ and R₅ are independently alkyl, alkenyl, alkynyl,branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridgedcycloalkyl, cycloalkenyl or cycloalkynyl radicals of C1 to C20 chainlengths, or any protecting group described in the book "ProtectingGroups in Organic Synthesis" by Theodora Greene and Peter G. M. Wuts;

where W₁ and W₂ are P(--OR)(with R being phenyl, phenylmethyl, ornegatively-charged oxygen), S═O, carbon, or sulfur, provided that if W₁is not P(--OR) W₂ is P(--OR) and provided that if J is a furanose orpyranose radical then W₁ is P(--OR),

A preferred subgroup of the above-described Class III of phosphocholinederivatives have the following structures: ##STR6## where R₁ is phenylor phenylmethyl, hydrogen, or nil; R₂ is hydrogen, phenylmethyl, or anyprotecting group described in the book "Protecting Group in OrganicSynthesis" by Theodora Green and Peter G. M. Wuts which can be cleavedby hydrogenolysis;

AA, BB, and Q are as defined above

where the central carbon atom of the three carbon unit is either the Roptical isomer, the S optical isomer, or any mixture of the two opticalisomers thereof;

Another preferred subgroup of the above-described Class III ofphosphocholine derivatives have the following structures: ##STR7## whereR₁ is phenyl or phenylmethyl, hydrogen, or nil; R₂ is hydrogen, phenylmethyl or any protecting group described in the book "Protecting Groupsin Organic Synthesis" by Theodora Greene and Peter G. M. Wuts which canbe cleaved by hydrogenolysis;

R₃ is hydrogen or a protecting group as described in the book"Protecting Groups in Organic Synthesis" by Theodora Greene and Peter G.M. Wuts.;

where the central carbon atom of the three carbon unit is either the Roptical isomer, the S optical isomer, or any mixture of the two opticalisomers thereof; and Q is defined above.

Still another preferred subgroup of the above-described Class III ofphosphocholine derivatives have the following structures: ##STR8## whereR₁ is phenyl or phenylmethyl, hydrogen, or nil; R₂ is a protecting groupas described in the book "Protecting Groups in Organic Synthesis" byTheodora Greene and Peter G. M. Wuts, or hydrogen if R₁ is not hydrogen;

and Q is defined above.

We have further found a novel, generally applicable method for thesynthesis of the above described broad classes of phosphocholinederivatives (Classes I, II and III).

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the FTIR spectrum of the composition comprising aphosphocholine derivative obtained from Irlbachia alata.

FIG. 2 is the proton NMR spectrum of the composition comprising aphosphocholine derivative obtained from Irlbachia alata in D₂ O at 400mHz.

FIG. 3 is the FAB/MB mass spectrum of the composition comprising aphosphocholine derivative obtained from Irlbachia alata.

5. DETAILED DESCRIPTION OF THE INVENTION

The glysosylated lysolecithins of the invention can be prepared bysynthetic methods or by enzymatic methods. The phosphocholinederivatives can be prepared either by synthetic methods or by methodsentailing extraction from plant materials.

5.1. Chemical Synthesis of Phosphocholine Derivatives

A wide variety of compounds having accessible alcoholic functionalitiescan be glycosylated following the classic Koenigs-Knorr methodology.Bochkov, A. F. and Zaikov. G. E., Chemistry of the O-Glycosidic Bond.Pergamon Press, 1979. As part of the synthetic route to phosphocholinederivatives with sugar, all but the anomeric hydroxyl group of the sugarto be introduced are protected either as esters or ethers, while theanomeric hydroxyl is being replaced by a halogen. The aglycon-sugarlinkage is then formed via alcoholysis. Finally, the protective groupsare selectively removed.

In the present invention, benzyl ethers or the benzilidine moiety areare the preferred protecting group, since they can be selectivelyremoved by catalytic hydrogenation, while leaving the sensitiveacyl-glycerol linkage intact. The glycosidation requires silver, mercury(Helferich modification), or cadmium salts as catalytic halogenabstractor, in the presence of a dehydrating agent (Timell, T. E.,Can.J. Chem. 1964, 42, 1456; Dejter-Juszynsky, M. and Flowers, H. M.,Carbohydr. Res. 1973, 30, 287; Marousek, V., Lucas, T. J., Wheat, P. E.,and Schuerch, C., Carbohydr. Res. 1978, 60, 85), and with or withoutauxiliaries such as crown-ethers. (Knochel, A. Ger, R., and Thiem, J.Tetrahedron Letters 1974, 551) More recent methodology makes use of thehalogen-abstracting power of non-nucleophilic bases such asdiisopropylethylamine and/or of molecular sieves in an anhydrous media.(Garegg, P. J. and Norberg, T., Carbohydr. Res. 1976, 52, 235) Thefollowing synthetic scheme is based on the latter reaction sequence:##STR9## The synthetic two-step scheme outlined above can be conductedwith commercially available materials.2,3,4,6-Tetrabenzyl-2,3-dibenzyl-4,6-benzylidene-glucose can beconverted into the 1-bromo- or 1-O-triflate compound by standardmethodology. Leroux, J. and Perlin, A. S. Carbohydr. Res. 1976, 47, C8.The corresponding phosphocholine derivatives are available throughAVANTI POLAR LIPIDS, Inc. All other reagents are available from ALDRICH.The methodology outlined above is also applicable to either 1-acyl or2-acyl (1-acyl detailed above).

5.2. Enzymatic Preparation of 1 or 2 glycosylated lysolecithins

As an alternative to the synthetic sequence outlined above, an in vitroenzymatic glycosidation simulating the biosynthetic process will producethe desired compounds in comparable yields. The natural glycosidationcatalysts are glycosyltransferases. These enzymes operate withuridinediphospho-glycosides (UDP-sugars) as substrates and ATP as theenergy source. While the enzymes have to be prepared from fresh plantmaterial, UDP-sugars, ATP, as well as the respective phosphocholinederivatives are commercially available. This synthesis has the advantageof being essentially a one-step process with the high selectivity andyields expected from an enzymatic reaction. The following schemedescribes the preparation of a glucoside. Other transferases, notspecific to glucose, could be applied in the preparation of glycosylatedlysolecithins with other sugars as well: ##STR10## 5.3. Total Synthesisof Phosphocholine Derivatives

A general synthetic method of synthesizing phosphocholine derivatives ofthe various structures described in section 3 is outlined as follows.

An alcohol is phosphorylated or glycosylated. The product issubsequently deprotected. The deprotected product is then alkylated oresterified to produce the phosphocholine derivatives. The general schemefor this outlined synthetic method is shown below. ##STR11## 5.4.Methods of Use

The phosphocholine derivative in Classes I, II and III are all useful intreating fungal infection by the administration to a warm-blooded animalof a therapeutically effective amount of a phosphocholine derivative.The pharmaceutical composition comprising the phosphocholine derivativeused for such administration may also contain pharmaceuticallyacceptable excipients and carriers.

Phosphocholine derivatives in Classes I and II are believed to be novelcompositions.

In order to treat a fungal infection, the antifungal agent of Classes I,II and III may be administered to a warm-blooded animal intravenously,intraperitoneally, subcutaneously, intramuscularly, orally, topically,by aerosol, or combinations thereof.

The antifungal agent of phosphocholine derivatives in Class II can beadministered intravenously in a range of about 0.1 to about 10 mg/kg.

The fungal agent of Class II can be administered intraperitoneally in arange of about 0.1 to about 10 mg/kg.

The fungal agent of Class II can be administered subeutaneously in arange of about 1 to about 20.

The fungal agent of Class II can be administered intramuscularly in arange of about 1 to about 20.

The fungal agent of Class II can be administered orally in a range ofabout 5.0 to about 30 mg/kg.

The fungal agent of Class II can be administered topically in a range ofabout 5.0 to about 15% by weight.

The fungal agent of Class II can be administered by aerosol in a rangeof about 5.0 to about 30 mg/kg/day.

The above dosage ranges may need to be doubled for those phosphocholinederivatives in Class I and III with lower antifungal activity which areidentical or similar to those in table 2 (see below).

6. Extraction of Phosphocholine Derivatives From Plants

Plants are not known to contain phosphocholine derivatives.

The general manner of chemical extraction from the plants can besummarized as follows.

The plant source material, such as the whole plant, the roots, leaves,stem and/or latex of the plant, is extracted with water and/or a watermiscible solvent. The preferred solvents are alcohol of 1-3 carbon atomsor acetone. The aqueous extract is extracted with butanol. Thebutanol-soluble fraction is subjected to gel filtration (e.g., overSephadex), reversed-phase column chromatography (e.g., C-8), orgel-permeation chromatography (e.g., divinyl benzene cross-linked gels)such as PL-GEL or membranes (e.g., an Amicon membrane) using water orwater and a water miscible solvent, with or without a buffer, as themobile phase. The water miscible solvent is preferably a 1-3 carbonalcohol, acetone or acetonitrile.

The useful phosphocholine derivatives containing compound is thefraction detected by NMR spectroscopy.

A specific member of the class of phosphocholine derivatives of thepresent invention is 2-palmitoyl-1-O-glycopyranosyllysolecithin shownbelow: ##STR12##

We have found that 2-palmitoyl-1-0-glucopyranosyllysolecithin is arelatively active antifungal agent similar in activity toL-a-Lysophosphatidyl inositol, discussed in Table 2 below.

We have found that one of the most active antifungal compounds has thefollowing structure. ##STR13## 6.1. Extraction

We have isolated by chemical extraction 1,22-docosan diolbisphosphocholine ester, the active antifungal compound contained in theplant Irlbachia alata. The leaves of Irlbachia alata were milled and 200g of the milled leaves was extracted with 1L ofdichloromethane/isopropanol (1:1 v/v) at room temperature for 24 hours.The extracted material was separated from the marc (i.e., residual ofthe plant after solvent extraction) and discarded. The marc was thenextracted with 1.5L of isopropanol/water (1:1 v/v) at room temperaturefor 24 hours. The marc was separated from the extract and discarded. Theisopropanol/water (1:1 v/v) soluble extract was partitioned betweenwater and ethyl acetate. The ethyl acetate phase was separated anddiscarded. The water soluble phase, after extraction with n-butanol, wasthen discarded. The n-butanol phase was subjected to filtration over twoSephadex LH-20 gel columns using 90% aqueous ethanol (for firstfiltration) and 20% aqueous acetone (for second filtration) as themobile phases. 1,22-docosandiol bisphosphocholine ester was collectedfrom the early fractions of each gel filtration.

We believe that several related genera are the same and/or closelyrelated to the genus Irlbachia, and would have similar medicinalproperties. One species from a closely related genus, Lisianthus nigrensis used in Mexico. The leaves are applied as a poultice to treat fungalinfections of the skin, feet, ankles and hands. A decoction of the rootis also taken orally as a "bitter" and as a febrifuge. Another speciesLisianthus alatus is considered to be the same as Irlbachia alata.Another species and genus of interest is Chelonanthus alatus. There areseveral uses described for Chelonanthus alatus, including oraldecoctions to treat smallpox, fevers and for gastric disturbances.

6.2. Spectral Characteristics

The isolated phosphocholine derivative fraction containing1,22-docosandiol bisphosphocholine ester has the characteristic IR,proton NMR and FAB mass spectra shown in FIGS. 1, 2 and 3, respectively.

The IR spectrum has peaks at approximately 1060, 1220, 1475, 1600-1700,2850, 2950 and 3400 cm⁻¹.

The ¹ H NMR spectrum has major peaks at δ 1.2, 1.4, 1.7, 3.1, 3.5, 3.7and 4.3.

The FAB/MB mass spectrum has major peaks (>40%) at m/z 657, 612, 587,586, 555, 493, 491, 475, 403, 277, 233, 201, 194, 179, 168, 165 and 163.

The high resolution mass spectrum (FAB⁺) has a molecular ion at 673.4669amu.

6.3. Total Synthesis of 2-palmitoyl-1-O-glucopyranosyllysolecithinExperimental Section

General

Tetrahydrofuran (THF) was distilled from potassium/benzophenone;benzene, triethylamine, and methylene chloride, N-methylmorpholine, andbenzyl alcohol were distilled from calcium hydride;2-bromoethylphosphorodichloridate was prepared according to theprocedure reported by Baumann et al Lipids, 17, 453 (1982) and wasfreshly distilled prior to use; trifluromethanesulfonic anhydride wasfreshly distilled under inert atmosphere; O-α-D-(Glucopyranosyl)trichloroacetimidate was prepared by the method of Schmidt. (a) R. R.Schmidt, J. Michael, Angew. Chem. Int. Ed Engl. (1980), 19, 731; (b) R.R. Schnmidt, J. Michael, Tetrahedron Lett. (1984), 25, 821. Anhydrousdimethylformamide (DMF) was obtained from Aldrich.S-(+)-1,2-O-isopropylidene glycerol and R-(-)-1,2-O-isopropylideneglycerol were obtained from Lancaster.2,3,4,6-Tetra-O-benzyl-D-glucopyranose was obtained from Sigma.Preparative thin layer chromatography plates was performed on Whatman2000 μTLC silica gel plates. Flash column chromatography was performedon Whatman 230-400 mesh silica gel using nitrogen pressure. ¹ H and ¹³ CNMR were provided by using a Varian 400 MHz spectrometer with chloroformas an internal reference unless otherwise noted. NMR shifts wereexpressed in ppm downfield from internal tetramethylsilane. Carbon 13multiplicities as determined by DEPT experiments are reported inparentheses following the chemical shift value according to thefollowing format: (0) for quaternary carbon, (1) for methine carbon, (2)for methylene carbon, and (3) for methyl carbons. NMR assignments weredetermined on the basis of COSY, HMQC, and HMBC and DEPT experimentsperformed on selected intermediates. NMR coupling constants are reportedin Hertz. Melting points were determined using a Buchi model 535 meltingpoint apparatus and are uncorrected.

The synthetic routes for the total synthesis of2-palmitoyl-1-O-glucopyranosyllysolecithin are outlined in the followingdiagrams and detailed in the subsequent discussion that refer to thesediagrams. ##STR14## (R)2,3-O-Isopropylidene-1-O-trifluromethylsulfonyl--glycerol.

A nitrogen-purged 250-mL three-necked roundbottomed flask fitted with athermometer, stopper, and septum was charged withS-(+)-1,2-O-isopropylidene glycerol (1.0 g, 7.6 mmol) dissolved inbenzene (75 ml). Triethylamine (1.25 mL, 9.0 mmol) was injected into thesolution, and the reaction mixture was chilled until a cloudy solutionappeared. Trifluoromethanesulfonic anhydride (1.25 mL, 7.6 mmol) wasthen added, and the reaction was stirred for 30 minutes with thetemperature maintained at 5° C. The solution was then filtered through abed of silica. The filtrate was concentrated under reduced pressure at30° C. to give an orange/brown oil (1.84 g, 7.0 mmol) in 92% yield whichwas used directly for the next step.

(2R)1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2,3-O-isopropylidene!glycerol1

2,3,4,6-Tetra-O-benzyl-D-glucopyranose (100 g, 0.182 mol) was dissolvedin THF (1.4 L) and chilled to -10° C. in a nitrogen-purged 3-Lthree-necked morton flask fitted with a thermometer, stopper, andmechanical stirrer. Sodium hydride 60% in oil (16.1 g, 0.403 mol) wasadded in 4 increments over 10 minutes, and the solution was stirred for30 minutes. (R) 2,3-O-Isopropylidene-1-O-trifluoromethylsulfonylglycerol(60.0 g, 0.227 mol) dissolved in THF (500 mL) was then dropped via anaddition funnel into the reaction mixture over a 30 minute period. Thesolution was stirred at -10° C. for 7 hours. Methanol (200 mL) was addeddropwise to quench excess sodium hydride, the resulting brown solutionwas rotary evaporated under reduced pressure and then the residueredissolved in chloroform (750 mL). The organic layer was washed withwater (2×750 mL). The combined aqueous layers were washed withchloroform (3×500 mL). Organic layers were pooled and rotary evaporatedunder reduced pressure to give a white solid which contained both α andβ-epimers of the desired product. The solid was triturated with diethylether to give a white solid of purely β--product and a mother liquorwhich contained α and β-epimers. The mother liquor was concentrated andflash chromatographed (silica gel, 20% ethyl acetate/hexane). Yield ofthe solid white β-epimer product (81 g, 0.123 mol) was 68%, mp 91°-91.7°C. (lit 83°-84° C.);¹ H-NMR (CDCl₃) δ7.4-7.29 (m, 18H), 7.20 (m, 2H),4.96 (d, 2H J=10.8), 4.84 (t, 2H, J=10.8), 4.75 (d, 1H, J=10.8), 4.65(d, 1H, J=12.4), 4.6-4.54 (overlapping dd, 2H, J=12H, J=10.4), 4.46 (d,1H, J=7.2, H₁ '), 4.38 (p, 1H, H₂), 4.12-4.02 (m, 2H, H_(1a), H₂), 3.89(pseudo t, 1H, J=7.2, H_(1b)), 3.79-3.6 (m, 5H), 3.50 (pseudo t, 2H),1.46 (s, 3H), 1.40 (s, 3H); ¹³ C-NMR (CDCl₃) δ138.529 (0), 138.370 (0),138.066 (0), 138.013 (O), 128.432, 128.409, 128.129 ,128.015, 127.901,127.810, 127.734, 127.666, 109.399 (0), 103.824 (C₁ '), 84-631 (C₃ '),82.120 (C₂ '), 77.713 (C₄ '), 75.748 (2), 75.058 (2), 74.891, 74.853,74.315 (2), 73.495 (C₁), 70.317 (2), 68.762 (C₆ '), 66.896 (C₃), 26.880(3), 25.386(3). Yield of the colorless, oily α-epimer (23 g, 0.035 mol)was 19%; ¹ H NMR (CDCI₃) δ7.4-7.24 (m, 18H), 7.14 (m, 2H), 4.98 (d, 1H,J=10.8), 4.88-4.78 (m, 3H), 4.67 (d, 1H, J=12), 4.62 (d, 1H, J=11.6),4.47 (d, 2H J=11.6), 4.37 (t, 1H, J=6.4), 4.07 (pseudo pentet, 1H), 3.96(t, 1H, J=8.8), 3.8-3.54 (m, 9H), 1.43 (s, 3H), 1.37 (s, 3H); ¹³ C NMR(CDCl₃) δ138.764 (0), 138.203 (0), 138.165 (0), 137.816 (0), 128.440,128.387, 128.364, 128.030, 127.947, 127.916, 127.886, 127.696, 127.590,109.422 (0), 97.482(C₁ '), 81.885(1), 79.890 (1), 77.508 (1), 75.703(2), 75.088 (2), 74.535 (1), 73.457 (2), 73.108 (2), 70.279 (1), 69.020(2), 68.314 (2), 67.040 (2), 26.827 (3), 25.424 (3).

(2R) 1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-glycerol 2.

A 5-L three-necked morton flask fitted with a mechanical stirrer,condenser, and stopper was charged with compound 1 (50 g, 76.2 mmol) in60% aqueous acetic acid (2.5 L). The acidic solution was refluxed for1.5 hours at 103° C. and then cooled to room temperature. Distilledwater (1.5 L) was added to the solution causing precipitation of a whitesolid. The acidic solution was extracted with methylene chloride (4×1 L)which was subsequently neutralized with sodium bicarbonate solution andconcentrated to a white solid. Trituration with diethyl ether gave whiteproduct. The remaining mother liquor was flash chromatographed (silicagel, 50% ethyl acetate/hexane) to give white solid product. The combinedyield (61.9 g, 0.101 mol) was 83%, mp 101.5°-102.4° C. (lit 76°-78° C.);¹ H NMR (CDCl₃) δ0.40-7.26 (m, 18H), 7.19 (t, J=3.5, 2H), 5.0-4.7 (m,5H), 4.64-4.5 (m, 3H), 4.46 (d, 1H, J=8.0, H₁ '), 4.0-3.60 (m, 11H, H₁'s, H₂, H₃, H₃ ', H_(6b) ', H_(6a) ', H₄ ', H₅ ', H₂, '), 2.55 (s, 2H,OH's); ¹³ C NMR (CDCl3) 38.529 (0), 138.332 (0), 137.952 (0), 137.740(0), 128.531, 128.550, 128.478, 128.189, 128.114, 128.091, 127.931,127.871, 127.749, 104.279 (C₁ '), 84.654 (C₃ '), 82.158 (C₂ '), 77.819(C₄ '), 75.779 (2), 75.081 (2), 75.028 (2), 74.527 (C₅ '), 73.571 (2),72.207 (C₁), 71.204 (C₂) 68.883, (C₆ '), 63.353 (C3).

(2S)1-O-(2113,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-3-O-tert-butyldimethylsilyl!glycerol 3

In a nitrogen-purged 100-mL round-bottomed flask fitted with a septumwas dissolved diol 2 (9.0 g, 14.7 mmol), imidazole (2.05 g, 30.2 mmol),and t-butyl dimethylsilylchloride (2.28 g, 15.1 mmol) in anhyd DMF (45mL). The reaction mixture was stirred under nitrogen for 2.5 days,transferred to a 1-L separatory funnel, and methylene chloride (250 mL)and water (250 mL) were added. The aqueous layer was extracted withmethylene chloride (2×250 mL) and then the combined organic layers werewashed with water (2×100 mL). After drying and concentration,purification by flash chromatography (silica gel, 33% ethylacetate/hexane) gave a colorless oil (9.2 g, 12.6 mmol) in 88% yield; ¹H NMR (CDCl₃) δ7.48-7.3 (m, 18H), 7.25-7.21 (m, 2H), 5.00 (d, 2H,J=11.2), 4.89 and 4.88 (overlapping doublets, 2H, J=10.8, J=10.4), 4.83(d, 1H, J=11.2), 4.67 (d, 1H, J=12.4), 4.60 and 4.59 (overlappingdoublets, 2H, J=12.4, J=10.8), 4.50 (d, 1H, J=7.6 H₁ '), 4.06-3.92 (m,2H), 3.9-3.62 (m, 7H), 3.6-3.52 (m, 2H), 3.04 (s, 1H, OH), 0.978 (s,9H), 0.142 (s, 6H); ¹³ C NMR (CDCl₃) δ188.552 (0), 138.385 (0), 138.005(0), 137.960 (0), 128.455, 128.440, 128.121, 128.060, 127.931, 127.863,127.772, 127.734, 127.696, 104.377 (C1'), 84.692 (C3'), 82.219 (C2'),77.804 (C4'), 75.771 (2), 75.073 (2), 74.959, (2), 74.717 (C5'), 73.541(2), 73.078 (2), 71.060 (C2), 68.785 (C6'), 63.998 (C3), 25.970 (3),18.668 (0), -5.299 (3).

(2S)1-O-(2,3,4,6-T6tra-O-benzyl-β-D-glucopyranosyl)-2-O-palmitoyl-3-O-t-butyldimethylsilyl!glycerol 4

A nitrogen purged 500-mL round-bottomed flask fitted with a septum wascharged with compound 3 (9.3 g, 12.8 mmol) and palmitic anhydride (6.94g, 14.0 mmol) in dry THF (200 mL). Dimethylaminopyridine (316 mg, 2.6mmol) and triethylamine (2.04 mL, 14.7 mmol) were added, and thereaction was stirred under nitrogen for 12 h. The mixture was thentransferred to a 2-L separatory funnel, and diethyl ether (500 mL) andwater (500 mL) were added. The aqueous layer was filtered throughWhatman No. 1 paper and extracted with diethyl ether (2×500 mL). Afterdrying over magnesium sulfate, the combined organic layers wereconcentrated and purifled by flash chromatography (silica gel, 14% ethylacetate/hexane) to give a light yellow oil (12.1 g, 12.5 mmol) in 97%yield; ¹ H NMR (CDCl₃) δ7.40 (br. s, 20H), 5.15 (5, 1H), 4.98 (t, 2H),4.84 (t, 2H), 4.76 (d, 1H), 4.67 (d, 1H), 4.59 (dd, 2H), 4.52 (d, 1H),4.13(dd, 1H), 3.84 (m, 6H), 3.67 (dd, 2H), 3.49 (t, 2H), 2.32 (t, 2H),1.61 (m, 2H), 1.25 (br. s, 24H), 0.98 (s, 9H), 0.97 (s, 3H), 0.14 (s,6H). ¹³ C NMR (CDCl₃) δ73.280, 138.597, 138.438, 138.127, 138.096,128.379, 128.356, 128.333, 128.083, 127.977, 127.863, 127.780, 127.605,127.582, 103.831, 84.556, 81.984, 77.721, 75.695, 75.020, 74.876,74.603, 73.488, 72.904, 68.754, 67.821, 61.661, 34.428, 33.950, 31.941,29.717, 29.687, 29.649, 29.619, 29.497, 29.459, 29.384, 29.300, 29.148,25.826, 24.953, 22.716, 18.268, 14.159, -5.375.

(2R) 1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-Opalmitoyl!glycerol 5

Procedure A Compound 4 (34.0 g, 35.1 mmol) was dissolved in THF (1.4 L)in a 3-L three-necked Norton flask fitted with a mechanical stirrer,thermometer, and a 500-mL addition funnel. The solution was chilled to0° C., and a solution of tetrabutylammonium fluoride (TBAF)(520 mL, 1.0Min THF) which was buffered to pH=6.5 with acetic acid was added dropwisethrough the addition funnel. The reaction mixture was stirred for 11 hat 0° C., left to sit at -15° C. for 12 h, and stirred again for 4 h atrt. Water (100 mL) was added, and the solution was concentrated to 200mL of solution. The concentrate was redissolved in methylene chloride(750 mL) in a 3-L separatory funnel and washed with water three times(750 mL, 2×500 mL). The combined aqueous layers were extracted withdiethyl ether (500 mL). The combined organic layers were concentrated togive a red oil which was purified by flash chromatography (silica gel,33-40% gradient of ethyl acetate/hexane). A white solid (28.0 g, 32.8mmol) was obtained in 93% yield. ¹ H NMR (CDCl₃) δ 7.36 (br. s, 20H),5.06 (t, 1H), 4.96 (dd, 2H), 4.84 (dd, 2H), 4.75 (d, 1H) 4.59 (m, 2H),4.53 (dd, 1H), 4.45 (dd, 1H), 4.14 (m, 2H), 3.91 (m, 2H), 3.78 (m, 6H),2.80 (s, 1H), 1.64 (m, 2H), 1.27 (br. s, 26H), 0.90 (t, 3H).

Procedure B Compound 4 (500 mg, 0.52 mmol) was dissolved in THF (20 mL)in a 100-mL three-necked round-bottomed flask fitted with two stoppersand a septum. Glacial acetic acid (9.5 mL) was added, and the solutionwas chilled to 0° C. A solution of TBAF (5.16 mL, 1.0M in THF) wassyringed into the chilled solution, and stinting was continued at 0° C.for 8 h and then at rt for 25 hours. Methylene chloride (50 mL) wasadded, and the entire solution was transferred to a 250-ml separatoryfunnel where it was neutralized with 1M disodium phosphate solution(2×75 mL). The combined organic layers were rotary evaporated underreduced pressure and the concentrate was purified by flashchromatography (silica gel, 25-40% gradient of ethyl acetate/hexane),yielding a colorless oil (424 mg, 0.497 mmol, 95%) which latersolidified upon standing; ¹ H NMR (CDCl₃) δ 67.36 (br. s, 20H), 5.06 (t,1H), 4.96 (dd, 2H), 4.84 (dd, 2H), 4.75 (d, 1H), 4.59 (m, 2H), 4.53 (dd,1H), 4.45 (dd, 1H), 4.14 (m, 2H), 3.91 (m, 2H), 3.78 (m, 6H), 2.80 (s,1H), 1.64 (m, 2H), 1.27 (br. s, 26H), 0.90 (t, 3H).

(2S) 1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-3-O-palmitoyl!glycerol 6

Compound 4 (3.0 g, 3.1 mmol) was dissolved in THF (120mL) in a 250-mLthree-necked round-bottomed flask fitted with a 60-mL addition funnel,glass stopper, and septum. TBAF (54 mL, 1.0M in THF) was added throughthe addition funnel over a 15 minute period. Glacial acetic acid (18 mL)measured in a graduated cylinder was then poured into the reactionmixture, and the solution was stirred for 45 minutes. The solution wasconcentrated under reduced pressure to approximately 30 mL of liquid andthen redissolved in methylene chloride (150 mL). The organic layer waswashed with water (3×120 mL) and neutralized with sodium bicarbonatesolution (2×150 mL). The combined aqueous layers were extracted withmethylene chloride (100 mL). The combined organic layers were dried overmagnesium sulfate, filtered, and concentrated. The resulting dark redconcentrate was purified by flash chromatography (silica gel, 25% ethylacetate/hexane) to give 6 a colorless oil which corresponded to an upperTLC spot (1.3 g, 1.52 mmol) in 46% yield. ¹ H NMR (CDCl₃) δ 7.36 (br. s,20H), 4.95 (m, 2H), 4.86 (m, 3H), 4.64 (d, 1H), 4.58 (m, 2H), 4.47 (d,1H), 4.16 (m, 1H), 3.96 (dd, 1H), 3.68 (m, 8H), 2.38 (t, 2H), 1.62 (m,2H), 1.27 (br. s, 24H), 0.96 (t, 3H). Isolation of a lower TLC spot gavea white solid (400 mg, 0.469 mmol) in 15% yield which corresponded tocompound 5; ¹ H NMR (CDCl₃) δ 7.36 (br- s, 20H), 5.06, (t, 1H), 4.96(dd, 2H), 4.84 (dd, 2H), 4.75 (d, 1H), 4.59 (m, 2H), 4.53 (dd, 1H), 4.45(dd, 1H), 4.14 (m, 2H), 3.91 (m, 2H), 3.78 (m, 6H), 2.80(s, 1H), 1.64(m, 2H), 1.27 (br. s, 26H), 0.90 (t, 3H).

Resilation of (2R) 1-O-(2,3,4,6-Tetra-O-benzyl-β-Dglucopyranosyl)-2-O-palmitoyl! glycerol 5

In a nitrogen-purged 50-mL round-bottomed flask fitted with a septum wasplaced compound 5 (318 mg, 0.373 mmol) dissolved in DMF (8 mL).tert-Butyl-dimethylsilyl chloride (281 mg, 1.86 mmol) and imidazole (254mg, 3.73 mmol) were added, and the solution was stirred for 22 h.Methylene chloride (50 mL) was added, and the reaction mixture wastransferred to a 250-mL separatory funnel. The organic layer was washedwith water (50 ml), and then the aqueous layer was extracted withmethylene chloride (2×50 mL). The pooled methylene chloride layers werewashed with water (2×75 mL), dried over magnesium sulfate, and thefiltered. The filtrate was concentrated and purifled by flashchromatography (silica gel, 14% ethyl acetate/hexane) to give 4 as ayellow oil (239 mg, 0.247 mmol) in 66% yield.

Resilation of (2S)1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-3-O-palmitoyl! glycerol6

In a nitrogen-purged 25-mL round-bottomed flask fitted with a septum wasplaced compound 6 (176 mg, 0.206 mmol) dissolved in anhyd DMF (5 mL).tert-Butyl-dimethylsilyl chloride (155 mg, 1.03 mmol) and imidazole (140mg, 2.06 mmol) were added, and the solution was stirred for 43 h.Methylene chloride (50 mL) was added, and the reaction mixture wastransferred to a 250-mL separatory funnel. The organic layer was washedwith water (50 mL). The aqueous layer was extracted with methylenechloride (2 ×50 ml). The methylene chloride layers were pooled methyleneand washed with water (2×75 mL), dried over magnesium sulfate, and thenfiltered. The filtrate was concentrated and flash chromatographed(silica gel, 14% ethyl acetate/hexane) to give 7 as a light yellow oil(190 mg, 0.223 mmol) in 95% yield; ¹ H NMR (CDCl₃) δ7.38 (br. s, 20H),4.99 (dd, 2H), 4.85 (t, 2H), 4.78 (d, 1H), 4.68 (d, 1H), 4.61 (dd, 2H),4.48 (d, 1H), 4.37 (d, 1H), 4.13 (s, 2H), 3.98 (m, 1H), 3.77 (m, 2H),3.67 (m, 3H), 3.52 (m, 2H) 2.35 (t, 2H), 1.67 (m, 2H), 1.31 (br. s,24H), 0.93 (s, 12H), 0.14 (s, 6H).

(2S) 1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-O-palmitoyl3-O-(2-bromoethyl)benzylphosphoryl! glycerol 15

Procedure A In a nitrogen-purged 100-mL three-necked round-bottomedflask fitted with two stoppers and a septum was dissolved freshlydistilled 2-bromoethylphosophorodichloridate (1.72 g, 7.11 mmol) indiethyl ether (20 mL). The solution was chilled to 0° C., andtriethylamine (8.15 mL, 58.5 mmol) was injected into the solution whichcaused precipitation of a white solid. A solution of compound 5 (1.0 g,1.17 mmol) in anhyd diethyl ether (55 ml) was injected into the chilledreaction mixture, and the ice bath was removed. The reaction was stirredfor 30 minutes after which benzyl alcohol (1.21 mL, 11.7 mmol) wasinjected into the reaction mixture. Stirring was continued at rt for 5d. The reaction was then filtered through a fritted glass funnel, andthe filtrate was concentrated. The orange concentrate was purified byflash chromatography (0-33% ethyl acetate/hexane) to give 15 as a lightyellow oil (566 mg, 0.501 mmol) in 43% yield; ¹ H NMR (CDCl₃) 67.38-7.25(br. s, 23H), 7.16 (m, 2H), 5.26 (m, 1H), 5.10 (t, 2H), 4.94 (m, 2H),4.81 (t, 3H), 4.71 (d, 1H), 4.61 (d, 1H), 4.55 (d, 2H), 4.39 (d, 1H),4.25 (m, 4H), 4.08 (dd, 1H), 3.73 (m, 3H), 3.64 (dd, 2H), 3.42 (m, 4H),2.27 (t, 2H), 1.58 (m, 2H), 1.25 (br. d, 24H), 0.89 (t, 3H).; ¹³ C NMR(CDCl₃) δ173.210, 138.559, 138.362, 138.096, 138.074, 128.667, 128.622,128.333, 128.318, 128.296, 127.962, 127.878, 127.757, 127.734, 127.696,127.605, 127.522, 103.862, 84.540, 81.969, 71.652, 75.589, 74.937,74.906, 74.686, 73.480, 70.469, 70.385, 69.680, 69.619, 68.777, 67.283,66.099, 66.069, 34.170, 31.887, 29.657, 29.619, 29.596, 29.452, 29.315,29.239, 29.080, 24,802, 22.647, 14.050.

Procedure B In a nitrogen-purged 100-mL three-necked roundbottomed flaskfitted with a thermometer, stopper, and septum was dissolved freshlydistilled 2-bromoethylphosphorodichloridate (1.42 g, 5.85 mmol) inmethylene chloride (15 mL). The solution was chilled to 0° C., andcompound 5 (1.0 g, 1.17 mmol) and a solution of N-methylmorphiline (1.28mL, 11.7 mmol) dissolved in methylene chloride (35 mL) was injected intothe solution over a 10 minute period. The reaction mixture was stirredat 0° C. for 5.5 h at which point a new TLC spot which co-spotted withsecondary alcohol 6 appeared. Stirring was continued for another 30minutes, and benzyl alcohol (1.21 ml, 11.7 mmol) was injected into thereaction. After 6 days of stirring, the reaction mixture was transferredto a 500-mL separatory funnel, and methylene chloride (150 mL) and water(200 ml) were added. The layers were separated, and the organic layerwas rotary evaporated under reduced pressure. The resulting oil wasflash chromatographed (silica gel, 33% ethyl acetate/hexane) to give 15as a yellow oil (250 mg, 19%); ¹ H NMR (CDCl₃) δ7.38 8-7.2 5 (br. s,23H), 7.16 (m, 2H), 5.26 (m, 1H), 5.10 (t, 2H), 4.94 (m, 2H), 4.81 (t,3H), 4.71 (d, 1H), 4.61 (d, 1H), 4.55 (d, 2H), 4.89 (d, 1H), 4.25 (m,4H), 4.08 (dd, 1H), 3.73 (m, 3H), 3.64 (dd, 2H), 3.42 (m, 4H), 2.27 (t,2H), 1.58 (m, 2H), 1.25 (br. d, 24H), 0.89 (t, 3H).; ¹³ C NMR (CDCl₃)δ173.210, 138.491, 138.286, 137.990, 137.975, 128.720, 128.652, 128.387,128.364, 128.015, 127.954, 127.878, 127.810, 127.780, 127.727, 127.681,127.613, 103.854, 84.495, 81.923, 77.781, 77.546, 75.688, 75.020,74.808, 74.747, 73.473, 70.438, 69.642, 68.633, 67.322, 66.759, 66.129,34.178. 31.925, 29.702, 29.664, 29.641, 29.490, 29.422, 29.368, 29.285,29.103, 24.802, 22.700, 14.198.

(2S)1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-O-palmitoyl-3-O-phosphatidylcholine!glycerol 16

A 45 mL Parr bomb equipped with a magnetic stirring bar was charged witha solution of phosphate 15 in toluene (10 mL). Condensed anhydroustrimethylamine (12 mL) was added quickly in one portion, and then thevessel was sealed and heated in an oil bath at 55° C. for 24 h. Thereaction mixture was concentrated to a viscous oil and triturated withethyl ether, upon which a white precipitate formed. the precipitate wasfiltered off, washed with ether, and then the combined etherealsolutions were concentrated to a glassy solid. Purification of thisresidue using preparative TLC (2000μ double elution with 75%,12.5%,12.5%methylene chloride/reethanol/hexanes gave inner salt 16 as a glassysolid;

(2S) β-D-glucopyranos-1-yl-2-O-palmitoyl-3-O-phosphatidylcholine!glycerol SP-19501

A solution of phosphatidylcholine 16 (200.4 mg, 0.197 mmol) in reagentgrade methanol (25 mL) was hydrogenated at 60 psi over 10% Pd/C (40 mg,20 wt %). After 30 h, the catalyst was ffltered off through celite andthe methanol washing were combined and concentrated. The residue wasdissolved in fresh methanol (25 mL) and resubjected to hydrogenation at60 psi over 80 mg (40 wt %) of 10% Pd/C. After 48 h, the reaction wasstill incomplete. After filtration, washing of the catalyst, andconcentration, the residue was subjected to hydrogenation using 400 mg(200 wt %) of Pd/C at 60 psi in methanol (25 mL). After 22 h, thecatalyst was filtered off through celite and the methanol filtrate andwashings were combined and concentrated to afford 92.8 mg (71.6%) of (S)SP-19501 as a white solid; ¹ H NMR (CD₃ OD) δ5.12 (br t, 0.5 H), 4.88(br m, 4.5 H), 4.25 (br m, 2H), 4.12-3.57(M, 12H), 3.4-3.1 (m containingsinglet at 3.18, 12H), 2.3 (m, 2H), 1.55(m, 2H), 1.24 (m, 22H), 0.86 (brt, 3H); ¹³ C NMR (CD₃ OD) δ 74.93, 104.80, 78.02, 77.93, 75.19, doubletat 71.53 and 71.49, doublet at 70.80 and 70.73, doublet at 67.79 and67.74, multiplet at 67.50, 62.53, doublet at 60.56 and 60.52, triplet at54.79, 34.88, 33.15, 30.85, 30.85, 30.66, 30.56, 30.46, 30.26, 26.10 and26.03, 23.82, 14.56; ³¹ P NMR (CD₃ OD) δ1.65.

(2S) 2,3-O-Isopropyiidene-1-O-trifluromethylsulfonyl-glycerol wasprepared according to the method described for the corresponding (R)isomer in 92% yield and used immediately.

(2S)1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2,3-O-isopropylidene!glycerol 8

2,3,4,6-Tetra-O-benzyl-D-glucopyranose (65 g, 0.12 mol) was dissolved inTHF (800 mL) and chilled to -10° C. in a nitrogen-purged 3-Lthree-necked morton flask fitted with a thermometer, stopper, andmechanical stirrer. Sodium hydride 60% in oil (33 g, 0.825 mol) wasadded in 4 increments over 10 minutes, and the solution was stirred for1 h. (S) 2,3-O-Isopropylidene-1-O-trifluoromethylsulfonylglycerol (0.15mol) dissolved in THF (200 mL) was then dropped via an addition funnelinto the reaction mixture over a 20 minute period at -10° to -15° C. Thesolution was stirred at -10° to -15° C. for 6 hours. The reactionmixture was filtered through a short plug of silica gel and concentratedto an orange brown oil, 114 g. Purification of the crude by flashchromatography using 50% ethyl ether/hexanes gave 39.8 g (67.6%) ofβepimer 8 as a white solid, along with 4 g (5.1%) of a mixture of α andβ epimers; mp of β anomer 85.7°-87.2° C.; ¹ H NMR of β epimer (CDCl₃) δ7.4-7.2 (m, 18H), 7.19-7.14 (m, 2H), 4.98-4.92 overlapping doublets at4.97 (J=10.8) and 4.94 (J=10.8), 2H!, 4.82 (t, 2H, J-10.8), 4.73 (d, 1H,J=10.4), 4.63 (d, IH, J=12.4), 4.58-4.51 overlapping doublets at 4.55(J=12) and 4.53 (J=10.8), 2H! 4.45 (dy 1H, J=7.2), 4.36 (p, 1H, H2),4.08 (pseudo triplet, 1H), 3.94-3.89 overlapping doublets at 3.92 (J=10)and 3.91 (J=9.6), 1H!, 3.82-3.57 (m, 6H), 3.47 (pseudo triplet, 2H),1.44 (s, 3H), 1.38 (s, 3H); ¹³ C NMR (CDCl₃) δ 138.569 (0), 138.384 (0),138.006 (0), 138.021 (0), 128.341, 128.258, 127.962, 127.856, 127.765,127.696, 127.620, 127.605, 109.467 (0), 103.869 (Cl'), 84.586 (C3'),82.075 (C2'), 77.705 (C4'), 75.680 (2), 75.005 (2), 74.815 (2 carbons,C2, C5'), 74.512 (1), 73.457 (C1), 71.151 (2), 68.785 (C6'), 67.017(C3), 26.895 (3), 25.393 (3).

(2S) 1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)--glycerol 9

A suspension of 8 (20 g, 30.5 mmol) in 60% acetic acid (800) was heatedto reflux for 1 h. Workup was similar to that described for the (R) diol2, providing 18 g (96% yield) of 9 as a white solid, which was ofsufficient purity after trituration with ether for the subsequent step.Diol 9 could be recrystallized from ether/hexane, mp 89.6-90.90C; ¹ HNMR (CDCl₃) δ7.38-7.27 (m, 18H), 7.16 (t, J=3.5, 2H), 4.98-4.74 (m, 5H),4.61-4.5 (m, 3H), 4.42 (d, 1H, J=8.0, H₁ '), 3.89-3.80 (m, 3H, H₁ 's,H2), 3.72-3.63 (m, 4H, H₃ H₃ ', H_(6b) '), 3.62-3.44 (m, 4H, H_(6a) ',H₄ ', H₅ ', H₂ '), 2.59 (s, 2H, OH's); ¹³ C NMR (CDCl₃) δ138.370 (0),138.119 (0),137.78 (0), 137.69 (0), 128.462, 128.447, 128.432, 128.060,128.038, 127.962, 127.894, 127.848, 127.810, 127.704, 104.195 (C₁ '),84.616 (C₃ '), 82.037 (C₂ '), 77.736 (C₄ '), 75.733 (2), 75.043 (2, 2carbons), 74.466 (C₅ '), 73.480 (2), 72.312 (C₁), 70.772 (C₂), 68.731,(C₆ '), 63.355 (C₃).

(2R)1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-3-O-tert-butyldimethylsilyl!glycerol 10

In a nitrogen-purged 100-mL round-bottomed flask fitted with a septumwas dissolved diol 9 (28.0 g, 45 mmol), imidazole (5.71 g, 90 mmol), andt-butyl dimethylsilylchloride (6.92 g, 45.3 mmol) in anhyd DMF (75 mL).The reaction mixture was stirred under nitrogen ovemight, transferred toa 1-L separatory funnel, and chloroform (300 mL) and water (300 mL) wereadded. The aqueous layer was extracted with chloroform (2×100 mL) andthen the combined organic layers were washed with water (3×100 mL).After drying (Na₂ SO₄) and concentration, purification by flashchromatography (silica gel, 50% ethyl ether/hexanes) gave 10 as acolorless oil (29.5 g) in 90% yield;

(2R)1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-Opalmitoyl-3-O-t-butyldimethylsilyl!glycerol 11

A mixture of 10 (22.2 g, 3 0.4 mmol), palmitic anhydride (16.5 g, 33.4mmol), dimethylaminopyridine (741 mg, 6.08 mmol), triethylamine (3.78 g,5.2 mL, 37.3 mmol) and anhyd THF (250 mL) was stirred under nitrogen atrt overnight. The mixture was poured into a 2-L separatory funnel,diluted with diethyl ether (500 mL) and water (500 mL), and the layersseparated. The aqueous layer was filtered through Whatman No. 1 paperand extracted with more diethyl ether (2×500 mL). The combined etherlayer was washed with water (3×200 mL) and then dried (MgSO₄). Followingfiltration, purification by flash chromatography (silica gel, 33% ethylether/hexane) gave 11 as a light yellow oil, 28.2 g, 96% yield);Compound 11 could be carried on to the next transformation withoutchromatographic purification.

(2S) 1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-Opalmitoyl!glycerol 12

Crude 11 (30.4 mmol based on 10) was dissolved in THF (100 mL) and thesolution was chilled to 0° C. A premixed solution of TBAF (520 mL, 1.0Min THF) which was buffered to pH=6.37 with acetic acid was addeddropwise via an addition funnel at 0° C. for 1 h, and then at -15° C.overnight. The reaction mixture was concentrated, water (100 mL) wasadded, and the resulting mixture was extracted with chloroform (3×300mL). The combined chloroform layer was washed with water (4×500 ML), andthen the combined aqueous layer was backextracted with diethyl ether(500 mL). After drying the combined organic layer over Na₂ SO₄,concentration gave a red oil which was purified by flash chromatography(50% ethyl ether/hexane). Evaporation of the product containingfractions afforded 12 as a white solid (24.6 g, 94.6% yield for twosteps);

(2R) 1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-3-Opalmitoyl!glycerol 13

Compound 11 (2.33 g, 2.41 mmol) was dissolved in THF (150 mL) in a250-mL three-necked round-bottomed flask fitted with a 60-mL additionfunnel, glass stopper, and septum. After cooling the solution to OoC,TBAF (24.1 mL, 1.0M in THF) was added through the addition funnel over a5 minute period. Glacial acetic acid (13.8 mL 241 mmol) was then pouredinto the reaction mixture to quench the reaction, and the resultingsolution was stirred for approximately 30 minutes. The reaction mixturewas poured into a separatory funnel containing ice water (500 mL) andmethylene chloride (200 mL). The layers were separated, and aqueouslayer was extracted twice more with methylene chloride (100 mL portions)and then the combined organic layer was washed with brine (400 mL).Following dring (MgSO4), filtration, and then concentration,purification by flash chromatography using 1/5 EtoAc/hexanes gavesecondary alcohol 13, 0.96 g (46.8%), as a colorless oil; Furtherelution gave 238 mg (11.6%) of primary alcohol 12; Also isolated was amixture of the two alcohols in 5.3% yield.

Resilation of (2S) 1-O-(2,3,4,6-Tetra-O-benzyl-β-Dglucopyranosyl)-2-O-palmitoyl! glycerol 12

The identity of 12 was established by resilylation of 12 according tothe procedure described above for the (R) isomer, compound 5.

(2R)1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-2-O-palmitoyl-3-O-(2-bromoethyl)benzylphosphoryl! glycerol 17

In a nitrogen-purged 1-L three-necked morton flask fitted with twostoppers and a septum was dissolved freshly distilled2-brorhoethylphosphorodichloridate (17.2 g, 71.1 mmol) in anhyd diethylether (500 mL). The solution was chilled to 0° C. and triethylamine(81.5 mL, 0.585 mol) was injected into the solution, causingprecipitation of a white solid. A solution of 12 (10.0 g, 11.7 mmol)dissolved in diethyl ether (250 mL) was cannulated into the mortonflask, and the solution was stirred for 1.5 hours. TLC showeddisappearance of 12. Benzyl alcohol (12.1 mL, 0.117 mol) was injectedinto the reaction mixture, and stirring was continued at rt for 16 h.The reaction mixture was then filtered through a fretted glass funnel.Filtrate was then concentrated and purified by flash chromatographytwice. First chromatography (silica gel, 33% ethyl acetate/hexane) andsecond chromatography (silica gel, 25% ethyl acetate/hexane) gave 17 asa light oil (5.5 g) in 42% yield;

(2S)1-O-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)-Opalmitoyl-3-O-phosphatidylcholinelglycerol 18

A 45 mL Parr bomb was equipped with a magnetic stir bar and then chargedwith a solution of 17 (1.17 g, 1.04 mmol) in benzene (15 mL). Anhydtrimethylamine (15 mL, 0.145 mmol) which had been condensed at -78° C.was quickly poured into the reaction vessel, and the bomb was sealed.The reaction was stirred at 55° C. in an oil bath for 24 hours behind ablast shield. The bomb vessel was then cooled to -78° C., opened, andleft in a hood to evaporate trimethylamine. The remaining solution wasrotary evaporated under reduced pressure, and the oily concentrate wasdissolved in methylene chloride and purified by preparative TLC (2000μ)-Double elution with 75%; 12.5%:12.5% methylene chloride/methanol/hexanegave inner salt 18 as an opaque glassy solid (223 mg, 21%). ¹ H-NMR(CDCl₃) δ0.32 (br. s, 20H), 5.21 (m, 1H), 4.90 (dd, 2H), 4.82 (m, H),4.64 (m, 2H), 4.50 (t, 2H), 4.42 (d, 1H), 4.22 (br. s, 3H), 3.95 (s,2H), 3.72 (s, 2H), 3.62 (t, 2H), 3.55 (s, IH), 3.40 (m, 4H), 3.10 (s,9H), 2.19 (m, 2 H), 1.47 (m, 2H), 1.20 (br. d, 24H), 0.87 (t, 3H). ¹³C-NMR (CDCl₃) δ73.393, 138.453, 138.377, 138.051, 137.998, 128.470,128.417, 128.356, 128.318, 128.235, 128.053, 128.007, 127.947, 127.856,127.780, 127.719, 127.636, 127.567, 103.899, 84.472, 81.984, 77.478,77.394, 77.311, 77.190, 75.672, 74.944, 74.550, 73.336, 68.663, 68.489,59.158, 59.135, 54.409, 54.349, 34.246, 31.902, 29.710, 29.664, 29.535,29.353, 29.330, 29.148, 24.7871 22.678, 14.121.

(2R) βD-glucopyranos-1-yl-2-O-palmitoyl-3-O-phosphatidylcholine!glycerol SP-19501

A solution of phosphatidylcholine 18 (130 mg, 0.127 mmol) in reagentgrade methanol (25 mL) was hydrogenated at 60 psi over 10% Pd/C (52 mg,40 wt %)- After 23 h, TLC showed an incomplete reaction- The catalystwas filtered off through celite and the methanol washings were combinedand concentrated- The residue was dissolved in fresh methanol (25 mL)and resubjected to hydrogenation at psi over 240 mg (185 wt %) of 10%Pd/C. After 20 h, the reaction was complete by TLC. The catalyst wasfiltered off through celite and the methanol filtrate and washings werecombined and concentrated to afford 64.0 mg (76.6%) of (R) SP-19501 as awhite solid; ¹ H NMR (CDCl₃) δ5.19 (m, 1H), 4.97 (s, OH+HDO), 4.34-4.26(br m, 2H), 4.16-3.95 (m, 3H), 3.9-3.6 (m, 6H), 3.42-3.14 (multipletcontaining singlet at 3.24, 13H), 2.37 (t, J=7.6, 2H), 1.62 (pseudo t,2H), 1.31 (m, 24H), 0.92 (t, J=7.2, 3H); ¹³ C NMR (CD₃ OD) δ175.02,104.88, 78.07, 78.04, 75.04, 72.97, 72.89, 71.52, 68.56, multiplet at67.50, doublet at 64.99 and 64.94, 62.65, doublet at 60.52 and 60.48,triplet at 54.74 (J=3.1), 35.14, 33.13, 30.85, 30.69, 30.54, 30.29,26.01, 23.79, 14.50; 31P NMR (CD₃ OD) δ1.35

(2R)1- Benzyl-(2'bromoethyl)-phosphproyl!-2,3-isopropylidene glycerol(19)

2-Bromoethylphosphodichloridate (20.0 g, 0.08 mol) was dissolved in CC14(50 ml) in a nitrogen-purged 0.5 L three-necked flask fitted with amagnetic stir bar, thermometer, and a 125-ml addition funnel. Thesolution was chilled to 0° C., and to this stirred solution was addeddropwise the solution of (S)-form solketal (10.7 g, 98 mol %) andN-methyl-morpholine (8.22 g,98 mol %) in CCL4 (25 ml). After 2 hours TLCshowed disappearance of solketal. To the reaction mixture was addeddropwise the solution of benzyl alcohol (44.6 g, 500 mol %) andN-methylmorpholine (8.38 g, 100 mol %). The reaction mixture was stirredunder nitrogen for 60 hours at room temperature. TLC showed the completereaction. The reaction mixture was filtered through Short filter #C, andthe solution was rotary evaporated to volume near 70 ml and purified byflash chromatography (silica gel, diethyl ether) to give colorless oil(15.1 g, 0.04 mol) in 45% yield; ¹ HNMR (CDCL3) δppm: 7.40 (br. s 5 H),5.2 (d, 2 H), 4.3 (br.s, 3 H), 4.0 (br.s, 3 H), 3.85 (br.s,1 H), 3.2 (s,2 H), 1.4 (d, 6H); ¹³ C NMR (CDCL3): 128.743, 128.682, 128.645, 128.114,128.076, 109.885, 77.364, 77.046, 76.727, 73.920, 73.837, 69.771,69.710, 67.760, 67.707, 67.654, 66.774, 66.721, 65.955, 29.353, 29.277,26.683, 25.204; ³¹ P NMR

(2R)1- Benzyl-(2'-bromoethyl)-phosphoroyl! 1-2,3-dihydroxy glycerol (20)

A nitrogen purged 1L roundbottomed flask fitted with septum was chargedwith compound 19 (19.5 g, 0.048 mol) in dry THF (50 ml) and the solutionof 1M H3PO4 (800 ml) was added. The reaction mixture was stirred undernitrogen by room temperature for 15 hours. TLC showed the completness ofthe reaction. Then the reaction mixture was transferred to a 2 Lsepapatory funnel. The acidic layer was extracted with ethyl acetate(7×450 ml). The combined organic extract was washed with water (2×850ml). After drying over sodium sulfate it was rotary evaporated and dryedin high vacuo for 10 hours to give a colorless oil (14 g, 0.04 mol %) in80% yield; ¹ H NMR (CDCl3), δppm: 7.38 (br.s, 5H), 5.2 (d, 2H), 4.25-3.8(multiplet, 6 H), 3.7-3.25 (br.m 5 H) ¹³ C NMR (CDCl3): 77.789, 77.774,77.349, 77.030, 76.712, 70.522, 70.491, 70.461, 70.431, 70.097, 70.044,68.898, 68.883, 68.822, 67.085, 67.032, 62.617, 62.496, 42.363, 42.280;³¹ P NMR (CDCl3): -0.485 (85% H3PO4).

(2R)1- Benzyl-(2'-bromoethyl)-phosphoroyl-2-hydroxy-3-O-triphenylmethylglycerol (21)

To a stirred solution of diol 20 (8.0 g, 21.6 mmol) in DMF (16 nil) wasadded diisopropylethylamine (4 ml, 105 mol %) followed by addition oftrityl chloride (6.4 g, 105 mol %). After 40 hours at room temperatureunder nitrogen the reaction was complete by TLC. The reaction mixturewas diluted twice with water and extracted with diethyl ether (4×100ml). The combined extract was dryed over sodium sulfate, concentratedand purified by flash chromatography silica gel, ethylacetate:hexane,1:1) to give 21 as a light oil 6.9 g (52.1%); ¹ H NMR(CDCl3) δ0.2-7.5 (br.m, 20 h), 5.07 (t, 2H), 4.12-4.26 (m,4H), 3.44 (dd2H), 2.05 (s, 1H), 1.26 (t, 1H). ¹³ C NMR (CDCl3): 138.772, 124.017,123.911, 123.820, 123.342, 123.266, 123-152, 122.462, 122.417, 72.593,72.274, 71.955, 65.105, 65.090, 65.030, 65.014, 64.954, 64.893, 62.109,62.056, 58.877, 24.680, 24.604. ³¹ P NMR (CDCl3) -0.158 (s).

(2R) 1- Benzyl-(2'-bromoethyl)-phosphoroyl-2-O-palmitoyl-3-O-triphenylmethyl glycerol (22)

To a stirred solution of compound 21 (6.9 g, 11.3 mmol) in dry THF (90ml) was added triethylamine (1.79 ml, 1 10,mol %), palmitic anhydride(6.13 g, 1 10 mol %) and dimethylaminopyridine (276 mg, 20 mol %). Thereaction was stirred under nitrogen for 3 hours untill TLC showeddisappearance of the starting material 21. The reaction mixture wasrotary evaporated to a small volume and purified by flash chromatography(silica gel, diethyl ether:hexane, 1:3 to elute UV-nonactive impurities,diethyl ether:hexane,1:1to elute compound 22). Yield 8.8 g (92.6%0,colorless oil, ¹ HNMR (CDCl3) 7.41-7.22 (m, 20 H), 5.20 (d, 1H), 5.04(t, 2H), 4.23 (m, 4H), 3.58 (s, 1H), 3.41 (s, 1H), 3.23 (s, 2H), 2.33(t, 2H), 1.62 (m, 3H), 1.24 (s, 24H), 0.88 (t, 3H). ¹³ C NMR 172.984,143.407, 143.285, 128.675, 128.607, 128.576, 128.523, 127.985, 127.848,127.180, 127.135, 86.672, 77.319, 77.000, 76.681, 70.901, 70.818,69.604, 69.581, 66.463, 66.440, 61.828, 34.284, 31.894, 29.672, 29.634,29.611, 29.437, 29.346, 29.285, 29.247, 29.232, 29.141, 29.095, 24.832,22.678, 14.121. ³² P NMR (CDCl3)-1.327.

(2R)1- Benzyl(2'- bromoethyl)-phosphoro!-yl-2-O-palmtoyl-3-hydroxyglycerol 23

Procedure A

To a stirred solution of compound 22 (3.2 g, 3.76 mmol) in 45 ml THF wasadded 45 ml 96% formic acid. After 2 hours qt room temperature thereaction was coplete by TLC. The reaction mixture was diluted twice withwater, neutralized with sodium bicarbonate (3×300 ml). The combinedextract was washed with water, dryed over sodium sulfate, rotaryevaporated to a small volume and purified by flash chromatography(silica gel, ethyl acetat: hexane, 1:3 to. elute less polar impurities,ethyl acetat:hexane,1:1to elute compound 23. Yield 1.65 g (72.7%),colorless oil.

Procedure B

A nitrogen purged 0.5 L round-bottomed flask fitted with condenser wascharged with compound 22 (1 g, 1.17 mmol) in dry benzene (230 ml) in thepresence of anhydrous CuSO4 (17.6 g). The reaction mixture was stirredat room temperature for 15 hours and then reflux for 2 hours untill thereaction was complete by TLC. The CuSO4 was filtered off through Shottfilter #C and concentrated in vacuo and purified by flash chromatography(silica gel, ethyl acetate/hexane, 1:1) to give a light yellow oil (0.47g, 0.77 mmol) in 66% yield. ¹ HNMR (CDCl₃): α7.40 (br. s, 5H), 5.2 (d,2H), 4.2 (mult, 8H), 2.32 (t, 1H), 1.62 (pseudo t, 2H), 1.31 (m, 24H),0.88 (t, 3H). ¹³ C NMR (CDCl3): 130.898, 123.872, 128.789, 128.698,128.538, 128.516, 128.114, 127.886, 126.968, 77.326, 77.008, 76.689,70.097, 70.074, 69.012, 68.951, 68.633, 68.604, 68.572, 68.542, 67.085,67.047, 67.032, 66.994, 65.272, 64.195, 62.731, 62.716, 34.041, 31.902,29.672, 29.588, 29.505, 29.444, 29.338, 29.239, 29.103, 24.749, 22.670,14.113. 31 P NMR: -3.069 (85% H3PO4).

(2R)-1-O-(2,3.4.6-Tetra-O-benzyl-β-D-gluco-pyranosyl)-2'-O-palmitoyl-3'-O-benzyl(2"-bromoethyl)- phosphoril!-glycerol (17)

To a stirred solution of O-(α-D-glucopyranosyl) trichloroacetimidate(24)(390 mg, 115 mol %) in dry methylene chloride (3 ml) was addeddropwise a solution of compound 6 (300 mg, 0.49 mmol) and borontrifluoride etherate (70 mg, 100 mol in dry methylene chloride (3 ml).The reaction mixture was stirred under nitrogen for 2 hours at roomtemperature, then more compound 24 (100 mg, 35 mol %) was added to bringthe reaction to the end. After 4 h, the reaction mixture was evaporatedto a small volume and separated by flash chromatography (silica gel,diethyl ether/hexane, 1:3) to give compound 17 as a colorless oil (120mg, 22%), which was identical to the material described earlier.

6.4. Antifungal Activity

The antifungal activity of the isolated phosphocholine fraction wasdetermined in vitro by using three fungal cultures--Candida albicans,Cryptococcus neoformans and Aspergillus fumigatus. The method used todetermine in vitro antifungal activity is discussed in McGinnis, M. R.,Laboratory Handbook of Medical Mycology, Academic Press, New York,London, p661 (1980); and Droughet E., Dupont, B., Improvisi, L., Vivian,M. A. and Tortorano, A. M., "Disc agar diffusion and microplateautomatized technics for in vitro evaluation of antifungal agents onyeast and sporulated pathogenic fungi" in In Vitro and In VivoEvaluation of Antifungal Agents, Eds. Iwata, K. and Vanden Bossche, H.,Elsevier Science Publishers, New York, Oxford p303 (1986).

The minimum inhibitory concentration (MIC) and the minimum fungicidalconcentration (MFC) are summarized in the table 1 below.

    ______________________________________                                        Fungus Culture MIC (ug/ml)                                                                             MFC (ug/ml)                                          ______________________________________                                        C. albicans    0.8                                                            C. neoformans  <0.1                                                           A. fumigatus   <0.1      <0.4-0.8                                             ______________________________________                                    

These results clearly indicate the significant antifungal activity ofthe isolated fraction containing against a variety of fungal cultures.

6.5. Antifungal Activities of the Phosphocholine Derivatives Class

A series of related analogs to2-palmitoyl-1-O-glucopyranosyllysolecithin obtained commercially fromAvanti Biolipids have also been found to have high antifungalactivities. A summary of the antifungal screening test is shown in table2. The analog compounds were tested for their activity against C.albicans, C. neoformans, A. fumigatus and T. rubrum. Partial inhibitionof the fungus of between 25 to 75% was measured along with the totalinhibition (MIC) by these anolog compounds. A description of the partialinhibition measurement can be found in R. L. Stiller, et al The Journalof Infectious Diseases, 147, No. 6 (1983). The structure of these analogcompounds is as follows. ##STR15## wherein R is the group identified intable 2.

                                      TABLE 2                                     __________________________________________________________________________    Test Results from Antifungal Screening Laboratory                                           MIC          Partial inhibition                                               (μg/ml)   (μg/ml)                                         Lecithin      CA CN AF  TR CA CN AF TR                                        __________________________________________________________________________    L-a-Lysophosphatidylcholine                                                                 63 16 >1000                                                                             63 16 n/a                                                                              31 31                                        Heptadecanoyl (C17:0)                                                         L-a-Lysophosphatidylethanol                                                                 >100                                                                             >100                                                                             >100                                                                              >100                                                                             >100                                                                             100                                                                              >100                                                                             >100                                      Amine, Oleoyl                                                                 (C18:1, cis!-9)                                                               L-a-Lysophosphatidylcholine                                                                 >500                                                                             500                                                                              >500                                                                              >500                                                                             250                                                                              250                                                                              >500                                                                             >500                                      Decanoyl (C:10)                                                               L-a-Lysophosphatidylcholine                                                                 500                                                                              125                                                                              125 250                                                                              n/a                                                                              31 n/a                                                                              63                                        Lauroyl (C12:0)                                                               L-a-Lysophosphatidylcholine                                                                 31 31 125 31 n/a                                                                              n/a                                                                              31 n/a                                       Myristoyl (C14:0)                                                             L-a-Lysophosphatidylcholine                                                                 >250                                                                             >250                                                                             >250                                                                              >250                                                                             >250                                                                             31 >250                                                                             >250                                      Stearoyl (C18:0)                                                              L-a-Lysophosphatidylcholine                                                                 31 31 63  31 n/a                                                                              n/a                                                                              31 n/a                                       Oleoyl (C18:1, Cis!-9)                                                        L-a-Lysophosphatidylcholine                                                                 31 31 63  500                                                                              n/a                                                                              n/a                                                                              31 63                                        Palmitoyl (C16:0)                                                             L-a-Lysophosphatidyl                                                                        >100                                                                             >100                                                                             100 >100                                                                             >100                                                                             100                                                                              n/a                                                                              >100                                      inositol                                                                      __________________________________________________________________________

6.6. Toxicity

The toxicity of the isolated phosphocholine derivative fraction is low,based on tests with Hep 2 cells indicating an ID₅₀ of greater than 1000ug/ml. The method used in determining cytotoxicity is discussed inMosmann, T.,"Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays", J. Immun.Methods, 65, 55-63, 1986.

The isolated fraction having the above-described in vitro antifungalactivity and low toxicity is expected to similarly exhibit significantin vivo antifungal activity against fungal infections which aredermatophytic, systemic, ophthalmic and vaginal. Other human and animalinfections treatable with the compounds of the present invention includeaspergilliosis, candidiasis, and cryptococcus infections.

It is expected that the same isolated fraction would be useful intreating fungal infestation in plants as well.

It is apparent that many modifications and variations of this inventionmay be made without departing from the spirit and scope thereof. Thespecific embodiments described are given by way of example only and theinvention is limited only by the terms of the appended claims.

A number of references are cited in the present specification, theentire disclosure of each of which is incorporated by reference herein,in its entirety.

What is claimed is:
 1. A composition comprising a bis-phosphocholinederivative obtained from Irlbachia alata characterized by:(a) IRspectrum having peaks at approximately 1060, 1220, 1475, 1600-1700,2850, 2950, and 3400 cm⁻¹ ; (b) ¹ H NMR spectrum having major peaks atδ1.2, 1.4, 1.7, 3.1, 3.5, 3.7 and 4.3; and (c) FAB/MB mass spectrumhaving major peaks (>40%) at m/z 657, 612, 587, 586, 555, 493, 491, 475,403, 277, 233, 201, 194, 179, 168, 165 and
 163. 2. The compositionaccording to claim 1 wherein the bis-phosphocholine derivative ischaracterized by an HRMS (FAB⁺) spectrum having a molecular ion at673.4669 amu.
 3. A composition comprising a bis-phosphocholinederivative obtained from Irlbachia alata by a method which comprises:(a)extracting the whole plant, the leaves, the stems, the roots or thelatex of the plant Irlbachia alata with a lower alcohol of about 1-3carbons, acetone, water or other water miscible solvent or combinationsthereof to obtain an aqueous soluble fraction; (b) subjecting theaqueous fraction to butanol extraction and the butanol soluble fractionto gel filtration using water or water and a water miscible solvent withor without a buffer as the mobile phase; or to reversed phase columnchromatography using water or water and a water miscible solvent as themobile-phase; or to gel permeation chromatography using water or waterand water miscible solvent and acetonitrile with or without a buffer asthe mobile phase; or combination thereof and (c) collecting thefractions detected by NMR spectroscopy.
 4. A pharmaceutical compositionwhich is useful in treating a fungal infection when administered to awarm-blooded animal comprising a therapeutically effective amount of anantifungal agent comprising a compound having the structure of:##STR16## where Q is C2 to C30 alkyl, alkenyl, alkynyl, branched alkyl,branched alkenyl, or branched alkynyl;Z is oxygen or sulfur; X and Y areindependently oxygen, sulfur, CH₂ or N-R₁ ; A, B, and T areindependently alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl,or branched alkynyl radicals of C1 to C20 chain lengths; or areindependently or together cycloalkyl or bridged cycloalkyl radicals ofring size C3 to C20, or cycloalkenyl radicals of ring size C4 to C20; Dis oxygen, sulfur, CH₂ or N-R₂ ; F is alkyl, alkenyl, alkynyl, branchedalkyl, branched alkenyl, branched alkynyl, cycloalkyl, bridgedcycloalkyl, cycloalkenyl or cycloalkynyl radicals containing C1 to C20carbon atoms; R₁ and R₂ are independently hydrogen, alkyl, alkenyl,alkynyl, branched alkyl, branched alkenyl, branched alkynyl, cycloalkyl,bridged cycloalkyl, or cycloalkenyl radicals containing C1 to C20 carbonatoms, or a protecting group; and a pharmaceutically acceptable carrier.5. A compound having the structure of: ##STR17## where Q is C2 to C30alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, or branchedalkynyl;Z is oxygen or sulfur; X and Y are independently oxygen, sulfur,CH₂ or N-R₁ ; A, B, and T are independently alkyl, alkenyl, alkynyl,branched alkyl, branched alkenyl, or branched alkynyl radicals of C1 toC20 chain lengths; or are independently or together cycloalkyl orbridged cycloalkyl radicals of ring size C3 to C20, or cylcoalkenylradicals of ring size C4 to C20; D is oxygen, sulfur, CH₂ or N-R₂ ; F isalkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branchedalkynyl, cycloalkyl, bridged cycloalkyl, cycloalkenyl or cycloalkynylradicals containing C1 to C20 carbon atoms; R₁ and R₂ are independentlyhydrogen, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl,branched alkynyl, cycloalkyl, bridged cycloalkyl, or cycloalkenylradicals containing C1 to C20 carbon atoms, or a protecting group. 6.The compound having the structure of: ##STR18##
 7. A method for treatinga fungal infection in a warm-blooded animal comprising administering tothe warm-blooded animal a therapeutically effective amount of thecomposition of claim
 4. 8. The method of claim 7, wherein thecomposition is administered intravenously, intraperitoneally,subcutaneously, intramuscularly, orally, topically, by aerosol ofcombinations thereof.
 9. The method of claim 8, wherein the compound isadministered intravenously in a range of about 0.1 to about 10 mg/kg.10. The method of claim 8, wherein the compound is administeredintraperitoneally in a range of about 0.1 to about 10 mg/kg.
 11. Themethod of claim 8, wherein the compound is administered subcutaneouslyin a range of about 1 to about 20 mg/kg.
 12. The method of claim 8,wherein the compound is administered intramuscularly in a range of about1 to about 20 mg/kg.
 13. The method of claim 8, wherein the compound isadministered orally in a range of about 5.0 to about 30 mg/kg.
 14. Themethod of claim 8, wherein the compound is administered topically in arange of about 5.0 to about 15% by weight.
 15. The method of claim 8,wherein the compound is administered by aerosol in a range of about 5.0to about 30 mg/kg/day.
 16. The composition of claim 4, wherein thecompound has the structure: ##STR19##